This project is primarily focused on functional studies of T cell receptor and NK receptor recognition. Our approaches in these areas revolve around: 1) the characterization of two monoclonal antibodies that recognize H-2Dd/P18-I10 peptide complexes, both in terms of their binding characteristics and their ability to block the in vivo priming of CTL responses; 2) the characterization of animals transgenic for a single chain MHC-I molecule, expressed in a b2 microglobulin negative background; 3) the development of bacteriophage display systems for the affinity maturation of expressed T cell receptors and for the identification of peptides that bind T cell receptors; and 4) the further refinement of expression systems for T cell receptors that preserve binding activity. With respect to monoclonal antibodies that bind the H-2Dd/P18-I10 complex, these were identified by Drs. K. Polakova and D. Plaksin in this laboratory and initial characterization included the demonstration of P18-I10 specific binding, evaluation of the fine specificity of these interactions and the comparison of the binding of these antibodies to these MHC peptide complexes with the binding of a cloned/expressed T cell receptor. The antibodies seem to be focused on the same region of the peptide as the TCR is, but are somewhat more tolerant of amino acid substitutions of the peptide. Although the analysis with synthetic peptides has clearly shown that the antibodies bind the decamer peptide bound to the MHC-I molecule, it has been very difficult to clearly demonstrate the presence of this complex as generated by the endogenous protein processing and presentation pathway. Attempts to visualize cell surface H-2Dd/P18-I10 complexes on the cell surface as generated from the endogenous expression of the HIV gp120 envelope protein in vaccinia vectors have been consistently negative, presumably due to a combination of the relatively low affinity of these antibodies and the low level of expression of the processed peptide. However, we have recently unequivocally demonstrated that mice given the specific mAb prior to in vivo immunization with the gp120 vaccinia vector, fail to mount a peptide specific H-2Dd-restricted response. In a dose dependent manner, the mAb blocks the primary immunization with the vaccinia expressed protein. This is indirect evidence that the priming MHC/peptide complex is the same as that recognized by the mAb. The characterization of the single chain H-2Dd transgenic mice on a b2-m negative background has revealed several important aspects of both T cell and NK cell recognition. The single chain H-2Dd molecule, expressed on a b2-m negative background, serves to positively select a diverse repertoire of CD8+ cells. These animals can mount a peptide specific response, indicating that they can adequately educate their T cells. In addition, antigen presenting cells from these animals clearly are capable of presenting H-2Dd-restricted peptides to specific T cells. In contrast to TCR recognition of the single chain H-2Dd molecule, NK recognition seems to be impaired. In particular, testing NK recognition by evaluation of the ability of bone marrow grafts to repopulate irradiated recipients, the single chain transgenics behave like b2-m negative animals, suggesting that the Ly-49A NK receptor is unable to physiologically interact with the single chain H-2Dd. In efforts to understand the contribution of particular amino acids of the MHC/peptide/T cell receptor interface to the affinity and specificity of binding, we have applied the general approach of antibody display libraries to a cloned T cell receptor. We now have expressed the TCR in bacteriophage and following random mutagenesis will select high and low affinity variant TCRs, sequence them, and characterize them further. Similarly, in collaboration with Dr. J. Scott, Simon Fraser University, we have identified several bacteriophage from a battery of peptide display libraries that encode novel peptides that bind the H-2Dd/P18-I10 restricted TCR. With a goal of assessing the nature of the TCR recognition that is involved in autoimmune diseases, in collaboration with Dr. E. Shevach?s laboratory, we have cloned the Va2 Vb2 TCR from a gastritis producing IAd restricted, H/K ATPase specific T cell clone, and expressed this as a three domain TCR. We are in the process of generating transgenic animals with this and related TCR, and are engineering the ATPase peptide/IAd ligand to allow detailed binding studies.